The activation of the platelet is important for the hemostatic process, since the platelet thereby becomes a full participant in the formation of lesion-directed aggregates, a secretor of vasoconstricting and mitogenic substances, and a surface for the generation of pro-coagulant protein factors. It is now well recognized that the hydrolysis of a quantitatively minor species of phospholipid, phosphoinositide (PI), by phosphoinositidase C (PIC) is one of the major initial events that couples a cell surface-directed agonist to a full cellular response. Another agonist-induced change in platelet PI metabolism described recently is phosphorylation of PI species by a 3-kinase (PI-3K). This reaction, in nucleated cells, appears to be required for mitogenic responses. The function of the resulting PI's is as yet unknown, especially in the anucleate platelet. Since PI-3K products are poor substrates for PIC, they probably function directly, rather than as precursors for signals. Major phospholipid, such as phosphatidylcholine (PtdCho), serves as a reserve for the thromboxane A2 precursor, arachidonic acid (C20:4), which is liberated in activated platelets via phospholipase A (PLA). All of these changes appear to be controlled by G-proteins. In the course of platelet activation, amorphous cytoplasmic precursors polymerize into organized cytoskeleton, and glycoproteins change their linkage to membrane cytoskeleton. We intend to explore how the metabolic changes in PI's catalyzed by PIC and PI-3K and the mobilization of C20:4 are coordinated with alterations in the platelet cytoskeletal apparatus, temporally and functionally. Four additional activatable platelet components: tyrosine kinase, the thiol protease calpain, G-protein(s), and the integrin GPIIb/IIIa, will be investigated with respect to PIC, PI-3K and PIA regulation. Techniques for monitoring platelet PIC and PI-3K by radioisotopic and mass analysis in conjunction with thin layer chromatography (TLC) and HPLC were pioneered, and are in common use in, this lab. We also have demonstrated expertise in measuring C20:4 mobilization and PLA activation by TLC and HPLC. These techniques, in part, will be applied to triton-insoluble cytoskeletal and membrane skeletal preparations as well as to immunoprecipitated phosphotyrosine-containing protein. The effects of two PI-3K products on gelsolin and profilin function (modulating actin polymerization) will be measured. Activation of tyrosine kinase, G-protein, and calpain will be monitored by immunoblotting techniques and their function, as well as that of GPIIb/IIIa, will be perturbed with appropriate inhibitors and agonists and compared with alterations in PIC, PI-3K, and PLA activities.